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1.
Mol Cancer Ther ; 17(6): 1240-1250, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29592883

RESUMO

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) in vitro After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control (P = 0.0159) and reduced tumor mitotic figures (P = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells in vitro We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma in vivo, thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. Mol Cancer Ther; 17(6); 1240-50. ©2018 AACR.


Assuntos
Antineoplásicos/farmacologia , Glioblastoma/metabolismo , Ferro/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organometálicos/farmacologia , Pironas/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Antineoplásicos/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glioblastoma/patologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Compostos Organometálicos/química , Pironas/química , Ratos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores
2.
J Pharmacol Exp Ther ; 322(3): 1228-36, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17600139

RESUMO

Clinical studies have shown gallium nitrate to have significant antitumor activity against non-Hodgkin's lymphoma and bladder cancer, thus indicating that gallium-based drugs have potential for further development as antineoplastic agents. In this study, we compared the cytotoxicity of gallium maltolate, a novel gallium compound, with gallium nitrate in lymphoma cell lines, including p53 variant and unique gallium nitrate-resistant cells. We found that gallium maltolate inhibited cell proliferation and induced apoptosis through the mitochondrial pathway at lower concentrations and more rapidly than gallium nitrate. Gallium maltolate produced an increase in intracellular reactive oxygen species (ROS) within 2 h of incubation with cells; this effect could be blocked by mitoquinone, a mitochondria-targeted antioxidant. The role of the transferrin receptor (TfR) in gallium maltolate's action was examined using monoclonal antibody (MoAb) 42/6 to block TfR function. However, although MoAb 42/6 reduced gallium maltolate-induced caspase-3 activity, it had only a minor effect on cell growth inhibition. Importantly, gallium maltolate induced apoptosis in cells resistant to gallium nitrate, and, unlike gallium nitrate, its cytotoxicity was not affected by cellular p53 status. Cellular gallium uptake was greater with gallium maltolate than with gallium nitrate. We conclude that gallium maltolate inhibits cell proliferation and induces apoptosis more efficiently than gallium nitrate. Gallium maltolate is incorporated into lymphoma cells to a greater extent than gallium nitrate via both TfR-independent and -dependent pathways; it has significant activity against gallium nitrate-resistant cells and acts independently of p53. Further studies to evaluate its antineoplastic activity in vivo are warranted.


Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gálio/farmacologia , Linfoma/tratamento farmacológico , Compostos Organometálicos/farmacologia , Pironas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação de Medicamentos , Humanos , Linfoma/patologia , Receptores da Transferrina , Proteína Supressora de Tumor p53
3.
Mol Cancer Ther ; 5(11): 2834-43, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17121930

RESUMO

Gallium nitrate is a metallodrug with clinical efficacy in non-Hodgkin's lymphoma. Its mechanisms of antineoplastic action are not fully understood. In the present study, we investigated the roles of transferrin receptor (TfR) targeting and apoptotic pathways in gallium-induced cell death. Although DoHH2 lymphoma cells displayed a 3-fold lower number of TfRs than CCRF-CEM lymphoma cells, they were 3- to 4-fold more sensitive to gallium nitrate. Despite a lower TfR expression, DoHH2 cells had greater TfR cycling and iron and gallium uptake than CCRF-CEM cells. In other lymphoma cell lines, TfR levels per se did not correlate with gallium sensitivity. Cells incubated with gallium nitrate showed morphologic changes of apoptosis, which were decreased by the caspase inhibitor Z-VAD-FMK and by a Bax-inhibitory peptide. Cells exposed to gallium nitrate released cytochrome c from mitochondria and displayed a dose-dependent increase in caspase-3 activity. An increase in active Bax levels without accompanying changes in Bcl-2 or Bcl-X(L) was seen in cells incubated with gallium nitrate. The endogenous expression of antiapoptotic Bcl-2 was greater in DoHH2 cells than in CCRF-CEM cells, suggesting that endogenous Bcl-2 levels do not correlate with cell sensitivity to gallium nitrate. Gallium-induced apoptosis was enhanced by the proteasome inhibitor bortezomib. Our results suggest that TfR function rather than TfR number is important in gallium targeting to cells and that apoptosis is triggered by gallium through the mitochondrial pathway by activating proapoptotic Bax. Our studies also suggest that the antineoplastic activity of combination gallium nitrate and bortezomib warrants further investigation.


Assuntos
Antineoplásicos/toxicidade , Gálio/toxicidade , Linfoma não Hodgkin/metabolismo , Mitocôndrias/metabolismo , Receptores da Transferrina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Inibidores de Caspase , Morte Celular , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Gálio/farmacologia , Humanos , Ferro/metabolismo , Células Jurkat , Linfoma não Hodgkin/enzimologia , Linfoma não Hodgkin/patologia , Complexo de Endopeptidases do Proteassoma/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/antagonistas & inibidores
4.
Int J Cancer ; 119(9): 2200-4, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16823846

RESUMO

The antineoplastic agent doxorubicin inhibits cell growth through mechanisms that include an interaction with iron, resulting in the generation of cytotoxic reactive oxygen species (ROS). Prior studies have shown that the wild-type hemochromatosis gene (wt HFE) may downregulate iron uptake and alter iron homeostasis in cells. We therefore tested the hypothesis that expression of wt HFE would affect the cytotoxicity of doxorubicin. Human breast cancer MCF-7 cells were transfected with an expression plasmid for a FLAG-tagged wt HFE gene [fwtHFE(+) cells], to examine the impact of wt HFE expression on doxorubicin-induced apoptosis. Our results show that, in MCF-7 cells, fwtHFE expression resulted in a reduction in cellular iron uptake and a decrease in the growth inhibitory effects of doxorubicin. Two micromolar doxorubicin inhibited the growth of fwtHFE(+) and fwtHFE(-) MCF-7 cells by 34% and 61%, respectively. In parallel, doxorubicin induced caspase-3-like activity in fwtHFE(-) cells, but not in fwtHFE(+) cells. On analysis with a DCF fluorescence assay, ROS could be detected in fwtHFE(-) cells but not in fwtHFE(+) cells exposed to doxorubicin. Western blot analysis of breast biopsy samples from patients revealed immunoreactive HFE and transferrin receptor proteins in both normal and malignant breast tissues. Our studies suggest that HFE expression and its consequent effect on cellular iron homeostasis may modulate doxorubicin-induced oxidative stress and apoptosis in breast cancer cells. Further investigation is warranted to determine whether HFE expression in tumor cells impacts on the clinical efficacy of doxorubicin.


Assuntos
Neoplasias da Mama/patologia , Doxorrubicina/toxicidade , Hemocromatose/genética , Apoptose , Caspase 3 , Caspases/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Ferro/metabolismo
5.
J Nucl Med ; 44(6): 943-6, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791823

RESUMO

UNLABELLED: Recent studies have revealed that the wild-type hemochromatosis protein (HFE) interacts with the transferrin receptor (TfR) and modulates TfR-mediated iron uptake by cells. Because of similarities in the transport of gallium and iron and the use of (67)Ga scanning in lymphoid malignancies, we examined the effect of HFE expression on (67)Ga uptake. METHODS: (67)Ga and (59)Fe uptakes were measured in HeLa cells transfected with a FLAG-tagged wild-type HFE (fHFE) gene under control of a tetracycline-repressible promoter. fHFE and TfR protein levels were measured by Western blotting; cellular transferrin (Tf) binding sites were measured by (125)I-Tf binding assay. RESULTS: Induction of fHFE expression produced an increase in TfR protein that was accompanied by a decrease, rather than an increase, in cellular (67)Ga and (59)Fe uptake. The difference in (67)Ga uptake between fHFE-expressing and fHFE-nonexpressing cells was markedly increased in the presence of Tf. Although fHFE expression produced an increase in cellular TfR protein, cell surface and intracellular Tf binding sites were actually decreased in these cells. CONCLUSION: Our studies suggest that expression of wild-type HFE in cells produces a decrease in (67)Ga uptake due to a reduction in available Tf binding sites for (67)Ga-Tf on the TfR. These results imply that (67)Ga uptake by cells with wild-type HFE may differ from cells with the HFE C282Y mutation.


Assuntos
Citratos/farmacocinética , Gálio/farmacocinética , Hemocromatose/genética , Hemocromatose/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Hemocromatose/diagnóstico por imagem , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Proteínas de Membrana/genética , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Transfecção
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